1. Field
This application relates to the art of analyzing samples for particular substances by means of fluorescent binding assays, and more particularly to apparatus, compositions and methods for such assays employing evanescent light.
2. State of the Art
Biosensor apparatus based on optical detection of analytes by fluorescence of tracer molecules, have attracted increasing attention in recent years. Such apparatus are useful for both diagnostic and research purposes. In particular, biosensors for a solid-phase fluoroimmunoassay, are becoming an important class of optical biosensor. A technique known as TIRF or total internal reflection, is one method for excitation/detection of fluorescence useful with biosensors for solid-phase assays.
In a typical such apparatus the biosensor is an optical substrate such as a fiber optic rod, to which is adsorbed or covalently bound a binding agent specific for a desired analyte. When the sensor is irradiated with light of an appropriate wavelength, the binding of the analyte to the immobilized binding agent results in a change (either a decrease or increase) in fluorescent emission of a tracer substance. The tracer substance may be the binding agent, the analyte, the complex, or a third, added tracer molecule.
It is desirable to have a system in which the desired sensitivity is achieved without requiring a xe2x80x9cwashxe2x80x9d step to remove unbound tracer before the fluorescence is measured. One approach to such a system has been to utilize evanescent light to selectively excite tracer molecules directly or indirectly bound to the immobilized binding agent. Evanescent light is light produced when a light beam traveling in a waveguide is totally internally reflected at the interface between the waveguide and a surrounding medium having a lower refractive index. A portion of the internally reflected light penetrates into the surrounding medium and constitutes the evanescent light field. The intensity of evanescent light drops off exponentially with distance from the waveguide surface.
In fluorescence assays using evanescent light, the waveguide is usually glass or a similar silica-based material, and the surrounding medium is an aqueous solution. The region of effective excitation by evanescent light in that situation is generally from about 1000 to about 2000 xc3x85 (angstroms). This region is sufficient to include most of the tracer molecules bound to the waveguide surface by means of interaction between the capture molecules and the analyte. However, the bulk of the unbound tracer molecules remaining in solution, will be outside the range of effective excitation and thus will not be stimulated to emit fluorescence.
Desirably, an immunofluorescent biosensor should be capable of detecting analyte molecules at concentrations of 10xe2x88x9212 or below. To date, most reports of evanescent-type biosensors indicate that at best, concentrations of 10xe2x88x9211 could be detected.
It is further desirable for speed and convenience in xe2x80x9croutinexe2x80x9d testing, for example testing of blood bank samples for viral antibodies, to have an evanescent immunofluorescent biosensor which is disposable and which provides multi-sample measurement capability. Multi-sample capability would allow a test sample and a control sample (such as a blank, or, for a competition-type assay, a sample preloaded with tracer molecules) to be simultaneously illuminated and measured. Simultaneous multi-sample capability would also speed up the process of analyzing multiple samples and would reduce the effects of variation in the level of exciting light which are known to occur with typical light source. However, in a typical prior art evanescent light device such as that of Block et al, the waveguide is a fiber optic rod whose shape makes it difficult to build a multi-well biosensor.
Another factor which affects the attainable sensitivity relates to the intensity of excitation light emitted from the waveguide. The intensity of fluorescence emitted by tracer molecules is in part dependent on the intensity of exciting light (which is the evanescent field). Therefore, increased evanescent light intensity should provide increased fluorescence which in turn would improve the detection sensitivity. The level of evanescent light is in turn dependent on the intensity of the light beam propagating in the waveguide and on the efficiency of reflection of light at the interface between the waveguide and the surrounding medium. The typical rod-shaped waveguides are not as effective in internal reflection as a design having two parallel surfaces would be.
Previous methods of immobilizing antibodies to optical substrates in evanescent biosensors also present some problems causing reduction in sensitivity. Many such methods utilize the xcex5-amino groups of lysine residues in the protein. This approach has at least two significant disadvantages due to the fact that most proteins have multiple lysine residues. First, the presence of multiple potential coupling sites (multiple lysine residues) results in multiple random orientations of antibodies on the substrate surface. If the substrate-coupled lysine residue is near the N-terminal of the antibody molecule, the antibody""s antigen binding site (which is near the N-terminal) may be effectively unavailable for binding of the analyte.
Second, if multiple lysines on the same antibody molecule are coupled to the substrate, the molecule may be subjected to conformational strains which distort the antigen binding site and alter its binding efficiency. For capture molecules immobilized by typical prior methods, generally only 20% or less of the binding sites are functional for analyte binding. Thus, it is desirable to have a site-specific method for coupling of the antibodies or other proteins, so that the capture molecules will be uniformly oriented and available for analyte binding.
Another problem relates to the levels of non-specific binding to the antibody-coated surface of the optical substrate. These levels are often sufficiently high to make detection of analyte at concentrations below about 10xe2x88x9210 molar (abbreviated M) very difficult. Nonspecific binding can be reduced by including a wash step after the sample is incubated with the coated substrate, to remove unbound tracer molecules. However, this is time-consuming and complicates the assay procedure. For convenience, a one-shot or homogeneous assay, that is, one which does not require a wash step, is much to be preferred. Second, non-specific binding can be a serious problem unless the surface is xe2x80x9cpassivatedxe2x80x9d with a masking agent such as bovine serum albumin or with a thin coating of hydrophilic polymer such as poly(ethylene glycol) or poly(methacrylate). Without such passivation (which introduces yet another step into the procedure), non-specific binding can be 50% or more of the specific binding. Even with passivated surfaces, non-specific binding can be sufficient to reduce detection sensitivity and reproducibility.
Thus, a need remains for an evanescent biosensor apparatus with improved sensitivity for detection of analytes at picomolar concentrations and below. A need further remains for an immunofluorescent assay and biosensor with properties of low non-specific binding and having uniformly oriented capture molecules.
The invention comprises a system including both apparatus and methods for an evanescent-light immunofluorescence assay capable of detecting sub-picomolar concentrations of analytes in solution.
The apparatus includes a biosensor comprising a planar waveguide having a plurality of immobilized capture molecules on at least one surface, the capture molecules being constructed to selectively bind a desired analyte. The waveguide surface forms one wall of at least one sample reservoir for holding a sample solution. A light source is included in the apparatus and operably arranged to focus light into the waveguide, where internal reflection within the waveguide results in the production of an evanescent light field which penetrates into the sample solution. In the assay, the test solution also contains a plurality of tracer molecules constructed to emit fluorescence upon stimulation by the evanescent light. The apparatus further includes. detection means for detecting fluorescence emitted by the tracer molecules, which is reflective of the amount of analyte bound to the capture molecules.
In a preferred embodiment, multiple wells or channels are provided on the surface of the waveguide, to permit simultaneous comparison of fluorescence from control and sample solutions.
In another preferred embodiment, a substantial portion of the surrounding edge of the waveguide is coated with a reflective coating to prevent light from escaping through the edge, thereby increasing the intensity of the evanescent field.
In another preferred embodiment, the biosensor has the capture molecules site-specifically immobilized such that the percentage of capture sites available is 50 to 75% or more of the number of immobilized capture molecules. In a further preferred embodiment, the waveguide coatings used to immobilize the capture molecules are selected to be resistant to non-specific protein binding.
The invention includes a method of immobilizing the capture molecules at a selected site on the molecule, so that the immobilized capture molecules are substantially uniformly oriented on the waveguide surface. In the method the waveguide surface is coated with a first coating having selected available reactive groups, and the capture molecules to be immobilized are treated to modify a single moiety on each capture molecule to produce activated capture molecules. The modified moiety is constructed to bind to the reactive groups of the first coating. The coated surface with the activated capture molecules under conditions to cause the modified moiety to couple to the first coating and thereby immobilize the activated capture molecules to the waveguide surface.
In one procedure, the waveguide is coated with avidin, and the capture molecules are conjugated to a biotin moiety which has a very strong affinity for avidin. In another procedure, the waveguide coating is a hydrogel film formed of polymethacryloylhydrazide treated to produce free maleimido groups, and the Fabxe2x80x2 capture molecules are oxidized to produce reactive thiol groups which can then be reacted with the maleimido groups. In a third embodiment, a silanized waveguide surface is further coated with polyethylene glycol derivatized with ethylenediamine groups. These groups are then reacted with oxidzed Fabxe2x80x2 capture molecules.
A particular embodiment of the invention is a sandwich-type assay capable of detecting sub-picomolar concentrations of human chorionic gonadotrophin. However, the coupling chemistry and apparatus described are useful for any type of fluoroimmunoassay for which the necessary binding reagents are available, including tests for serum antibodies to selected pathogens.